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Full text of "Some Enumerative Studies on Malarial Fever"

159 



Some Enumerative Studies on Malarial Fever. 
By Major Bonald Boss, F.B.S., and David Thomson, M.B., Ch.B., D.P.H. 

(Beceived October 12, — Bead December 8, 1910.) 

Prefatory Note by B. Boss. — Towards the end of last year the Advisory 
Committee for the Tropical Diseases Beseareh Fund (Colonial Office) allotted 
considerable funds to the Liverpool School of Tropical Medicine for 
researches to be carried out in Liverpool. This enabled us to commence, 
under my direction, a number of minute co-ordinated studies on cases 
admitted into the Tropical Ward of the Boyal Southern Hospital — material 
which, though it offered peculiar facilities for research, had long remained 
neglected owing to want of funds. The cases (occurring in the clinics of 
Dr. Macalister, Dr. Lloyd Boberts, and myself) were placed in charge of 
Dr. David Thomson ; the chemical studies in charge of Dr. G-. C. E. 
Simpson; the parasitological studies in charge of Dr. H. B. Fantham. 
Parallel researches on animals w T ere also assigned to Dr. John Thomson who 
is working under Sir Edward Durning-Lawrence's fund for the investigation 
of the effect of temperature on disease ; Dr. V. T. Korke (Beseareh Fellow) 
has studied coagulation times and other details ; the literature was in charge 
of Mr. W. B. Drawz, the Malaria Bibliographer (Advisory Committee's 
Fund); and much valuable help has been given by the staffs of the 
University, the School of Tropical Medicine, and the Boyal Southern 
Hospital of Liverpool, and by Sister Linaker of the Tropical Ward. 

The researches were commenced on January 1, 1910. A paper by 
Dr. David Thomson and myself, describing a regular periodical increase of 
the trypanosomes in a case of Sleeping Sickness, was published ;* and we 
now present to the Society brief accounts of our results regarding malaria, 
blackwater fever, trypanosomiasis, and various therapeutical agencies, 
obtained (mostly by new methods) up to the end of July, 1910. Further 
details will be published, if necessary, in the ' Annals of Tropical Medicine/ 
Liverpool. 

1. Preliminary. — For many years past little information which is both 
new and exact has been added to our knowledge of the pathology of malaria. 
This has probably been due to the exhaustion of the older methods of 
research, which, being purely qualitative, have failed to indicate the precise 
correlations between the numbers of the parasites present in a patient and the 
various pathological and therapeutical reactions. For example, out of 

* < Boy. Soc. Proc.,' B, July 21, 1910, vol. 82. 

N 2 



160 Major R. Ross and Dr. D. Thomson. [Oct. 12, 

51 and 38 successful inoculations of men by means of infected blood and 
infected Anophelines respectively, in not a single one has any exact 
estimate been given of the numbers of parasites inoculated, or recovered 
after the lapse of the incubation period; and, though many researches on 
quinine have been made, we know of none in which its direct effect upon 
the numbers of the parasites in the patient has been correctly measured. 
Moreover, the older methods often failed to reveal the parasites at all, unless 
they were present in large numbers. Hence our first care was to elaborate 
more exact methods both for detection and for enumeration. This was done 
early in the year (by E. E. and D. T.) — though we have by no means reached 
finality yet. The new methods were next employed for all the cases in the 
Tropical Ward, daily counts of the parasites and often of the leucocytes ?> 
together with other estimations, being made (by D. T.). At the same time 
parallel chemical studies were carried out (by G. C. E. S.) and therapeutical 
ones (by D. T.) ; and the measurements were carefully charted and compared. 
Even at this preliminary stage the results (which are given at the end of 
this paper) include more accurate verifications of some old conjectures,, 
and demonstrations of some new theorems. 

2. The - Detective Method Used. — In the ordinary methods the blood is 
spread out in a thin film, liquid or dry — 1 cu. mm. covering, say, 4 sq. cm. 
of area. If the field of the oil-immersion lens has a diameter of 0*165 mm.,, 
and can be thoroughly searched by the eye in three seconds, about 12 hours' 
work would be required to examine the whole of 1 cu. mm. of blood in this 
way ; and if the specimen contains only one parasite it may not, by bad 
luck, be found until nearly the whole of the area has been searched. We 
have, therefore, employed the " thick film process ' ; described by one of 
us (E. E.) in 1903. In this 1 cu. mm. of blood is spread out over only 
about one quarter of a square centimetre or less, and dried ; the haemoglobin- 
is then gently washed out with water; and the residue, consisting of 
parasites, leucocytes, platelets, and the stromata of the red corpuscles, is. 
carefully stained by any convenient method, with or without fixation. By 
this simple means the whole of 1 cu. mm. of blood can be searched in less 
than an hour ; but the identification of the Plasmodia requires considerable 
practice. 

3. The Enumerative Method Used. — The younger Plasmodia are too small 
to be counted by an ordinary haBmocytometer ; hence their number has 
usually been estimated by comparison with the number of red or white cells,, 
the latter figure being determined by the hasmocytometer. This gives 
a large compound error ; for if e is the percentage error made in counting, 
the red or white cells in one specimen, and e' is the percentage error made in 



1910.] Some Enumerative Studies on Malarial Fever. 161 

computing the proportion of parasites to each corpuscle in that specimen, 
then, by a simple calculation, the total percentage error will be e + e' + ee'flQO. 
(For example, if e = +5 per cent, and e' = + 10 per cent., the total percentage 
error will be +15*5 per cent.) 

Our method consists simply in making a measured quantity of blood into 
a thick film preparation and then counting all the parasites in it. The blood 
is measured by determining the diameter of a fine capillary tube under the 
microscope and then calculating and marking off the length of the tube 
required to hold the given quantity (as suggested by Dr. Wakelin Barratt). 
Thus a uniform tube of 0*180 mm. diameter and 4 cm. length will contain 
1'018 cu. mm. — a convenient size. The blood is sucked up into the tube and 
then quickly discharged upon the glass slide in one or several measured 
droplets, first samples being rejected. The dehsemoglobinised thick film is 
fixed before staining, in order to avoid loss, and the whole area is scrupulously 
searched with the aid of a sliding stage. 

Mr. M. Greenwood, Junior, informs us that if n is the number of parasites 
counted in the unit (say, 1 cu. mm.) of blood, and m is the number of such 
units in the patient's whole body, then the total number of parasites in him, 
assuming uniform distribution, will be mn + 0'67449m^/n. Thus the probable 
percentage error is 6*7 '4:4:9 /y/n, the factor m cancelling out from the ratio. 
This is important, because it shows that the error depends, not on the 
magnitude of the measured sample taken, but upon the number of parasites 
actually counted in it. Hence, in order to obtain results within an assigned 
error, we take a large sample when the parasites are scarce and a small one 
when they are numerous ; the number of parasites which we must find and 
count being given by the formula n =. 4550/g 2 , where e is the permissible 
error. 

The greatest error occurs when the parasites are so scarce that it is 
difficult to find enough of them to count up to the required standard ; or 
when they are so numerous that it is difficult to measure a volume of blood 
small enough to contain an easily countable number of them. In these 
preliminary researches we have seldom examined at one sitting more than 
1 cu. mm. of blood, or less than \ cu. mm. 

When the parasites were very numerous, we have sometimes fallen back 
on estimating their number by comparison with leucocytes, but are now 
elaborating improvements and special instrumen ts for such, and other, 
details. The method is also accurate and rapid for other blood parasites, and 
for leucocytes. Other sources of error are (1) inaccurate measurements of the 
quantity of blood used, and (2) difficulty of seeing or distinguishing badly 
stained parasites. 



162 Major K. Ross and Dr. D. Thomson. [Oct. 12, 

One kilogramme of blood of average density (1057*5) contains 945,626 
cu. mm., and the blood in a man's body is estimated to weigh about 4*9 per 
cent, of the total body weight — from which data it is easy to compute the 
total number of parasites in a patient. A man of 64*74 kilogrammes, or about 
10 stone, will contain about 3,000,000 cu. mm. of blood. 

4. The Cases studied by us numbered 33. All had been infected in West 
Africa, or America, so that none was of less than some weeks' duration when 
admitted into hospital in Liverpool. All were males of from 18 to 60 years 
of age. They were mostly shipmen or traders, and two were negroes. The 
parasites of all were counted almost every day and sometimes several times 
a day ; and the temperature was taken every four hours, or more often. One 
case showed Plasmodium malarias, together with crescents (sexual forms of 
P. falciparum). Eight showed P. vivax only, and 24 P. falciparum only, two 
of the latter containing only sexual forms. Quinine was often withheld for 
some days while other methods of treatment were used. The cases were 
studied for over 600 days altogether. There were no deaths. 

All the cases will be recorded in the 'Annals of Tropical Medicine,' 
vol. 4, No. 3, but charts of Cases 17 and 20 are given on pp. 164 and 165. 

At this preliminary stage of the enquiry we have undertaken to study only 
the gross daily correlations between the numbers of parasites and other 
phenomena — leaving more minute hourly or four-hourly analysis to future 
work. Hence, if a number of counts or other observations have been made 
on one day, we have recorded only the averages in the tables. For tempera- 
tures, however, it has been thought best to record maxima instead of averages, 
and, as the tables should be as economical as possible, we have used the 
hwmatothermic scale suggested by one of us (K. E.), ill which the 10° between 
95° and 105° F., or between 35° and 45° C, are divided into 100 parts. 
Except in Case 6, specially studied by Dr. Fantham, we have not distin- 
guished in the tables the various forms, sets, or stages of P. vivax — this 
being reserved for future study. Similarly, the treatment, leucocytes, 
haemoglobin, and urobilin are not differentiated too minutely. Case 25 was 
counted by Dr. Korke. 

. 5. The Correlation between the Parasites and the Fever. — It is well known 
that fever is caused only by the asexual forms of the parasites, but though 
these are generally thought to be more abundant during pyrexial periods, 
no extensive numerical studies on the point appear to have been made, 
with the result that some sceptics still profess doubts on the subject. Our 
cases, except four without fever (Cases 1, 15, 16, 27), would seem to indicate 
a very strong (almost convincing) correlation. Twenty-one of them suffered 
each from one pyrexial period, lasting from one to seven days, and preceded 



1910.] Some Enumerative Studies on Malarial Fever, 



163 



or followed by apyrexia, and in all, except Case 32, the asexual parasites 
were very much more numerous during the pyrexial period. Nine of the 
cases (7, 17., 18, 19, 20, 23, 24, 26, 32) suffered from one or two relapses 
each (13 relapses altogether), and every relapse, as well as every original 
attack, was associated with a comparatively large number of parasites 
(43 pyrexial periods altogether). Similarly, there were 46 apyrexial periods 
(including the four cases without fever), all associated with comparatively 
small numbers of asexual parasites, and conversely not a single marked rise 
in the number of asexual parasites took place without corresponding fever. 

For further detail we compare the numbers of parasites per cubic milli- 
metre counted on days of fever with those counted on days without fever. 
A febrile clay is taken as any one on which the patient's temperature exceeds 
98°*6 F. ( = 36° haematometric scale), or any one which comes between two 
tertian paroxysms (Cases 9, 14, 24). The non-febrile days on which no 
asexual forms could be found in 1 cu. mm. of blood are omitted. The results 
are — 



Cases. 



Days. 



Total 
parasites. 



Average per 
day. 



Ratio, 



P. vivax. 



Fever ... 
No fever 

Fever . . . 
jSTo fever 



8 
8 


30 

68 


136,062 

8,487 


4,535 
125 


P 


'. falcipanv, 


m. 




21 
19 


97 

83 


1,319,880 

38,277 


13,607 

461 



36-3 



29-6 



If we had not omitted the non-febrile days on which no parasites were 
found, the non-febrile daily averages would have been much smaller than 
the figures given above, and the ratio of the febrile averages to them much 
larger, so that the febrile excess is very marked. An examination of the 
details will further convince the reader of the great fall in temperature 
which accompanies the fall in the number of parasites. We should note 
that the comparatively small numbers of P. vivax found are due to the fact 
that the specimens generally contained a large proportion of mature 
parasites, not as yet disintegrated into spores, these of course being absent 
from peripheral blood containing P. falciparum, in which only spores or 
young forms generally occur. There is no reason for considering quinine 
in this connection, because it probably affects the fever only through its 
action on the parasites. 



164 



Major R. Ross and Dr. D. Thomson. 



Oct. 12, 









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166 



Major R. Ross and Dr. D. Thomson. 



[Oct. 12, 



6. The Degree of Fever Associated with Various Numbers of Parasites. — 
We know of no extensive and exact studies on this point, and ours are not 
yet complete. On 47 occasions the number of parasites was carefully 
compared with the maximum degree of temperature reached during the 
febrile paroxysm caused by them, and the following results were obtained: — 



No. of parasites 

Maximum temp., deg. H.P. 

~Eo. of parasites 

Maximum temp., deg. H.F. 



P. 


vivax (12 


! occasions). 






1500 


1400 


580 


440 


430 1 


260 


84 


101 


97 


102 


76 


76 


232 


150 


133 


100 


83 




46 


44 


34 


34 


20 
(rigor) 





256 
54 



(Coefficient of correlation), r = '68561±0 '10318. 



P. falciparum (35 occasions). 



No. of parasites 

Maximum temp., deg. H.P. 

No. of parasites 

Maximum temp., deg. H.F. 

No. of parasites 

Maximum temp., deg. H.P. 

No. of parasites 

Maximum temp., deg. H.P. 

No. of parasites 

Maximum temp., deg. H.F. 

No. of parasites 

Maximum temp., deg. H.P. 



303,000 

108 

36,000 

60 

16,000 

52 

7,500 

90 

5,200 

76 

2,500 

50 



58,000 


55,000 1 


82 


88 


34,000 


31,000 


86 


76 


15,000 


15,000 


54 


54 


7,400 


6,700 


40 


86 


4,000 


4,000 


70 


40 


2,000 


1,860 


46 


38 



54,000 

100 

26,000 

94 

15,000 

70 

6,500 

83 

3,600 

80 

1,000 

15 



50,000 

98 

26,000 

101 

9,700 

56 

6,000 

52 

3,000 

38 

200 

50 



45,000 

76 

25,000 

80 

9,000 

70 

5,800 

70 

2,600 

64 



(Coefficient of correlation), r = '60331 ±0 '07251. 



These figures, together with the numerous ones given in the table of cases > 
show a very marked correlation, but there are, of course, many minor 
deviations. In numbers of the specimens there were certainly two sets of 
parasites of different ages, which were often counted together, though only 
one set could have been concerned with the fever. It is probable also, 
though by no means certain, that the resistance to the toxin of the Plasmodia 
varies, not only in different persons, but in the same person at different 
stages in the course of his infection and under different physiological 
conditions. Our new methods will, we hope, enable us shortly to undertake 
very detailed studies on these points. 

7. The Pyrogenic Limit. — It is quite evident that if the parasites fall below 
a certain limit, they are no longer numerous enough to cause fever. Thus 
with P. vivax, an average of 125 parasites of all ages and forms counted 
during 68 days in 8 cases, and with P. falciparum an average of 460 asexual 
forms counted during 83 days in 19 cases, failed to produce fever. The actual 
limit above which they become pyrogenic probably varies in different cases. 
With P. vivax, as many as 1500, 852, and 540 were found in Cases 2, 6, and 7 



1910.] Some Enumerative Studies on Malarial Fever. 167 

without fever, and as few as 150 and 50 in Cases 5 and 9 with very slight 
fever (98°*6 F.), so that about 200 to 500 may perhaps be taken as the usual 
limit. With P. falciparum, as many as 1620 (asexual) were found as an 
average of four non-febrile days in Case 26, and 1196 as an average of six 
non-febrile days in Case 25 ; while in Case 18 from 16 to 1860 were present 
for 22 days without causing any marked fever, so that 600 to 1500 may 
perhaps be adopted as the usual limit. With P. malarim (Case 1), an 
average of 79 were present for 13 days without fever, but about 140 twice 
caused slight rigor. 

8. The Asexual Forms between the Relapses. — It is generally thought and 
taught that these forms tend to " disappear " between the relapses — though 
of course exceptions, especially in children, are noted — and to reappear 
during them ; and several hypotheses, such as that of parthenogenesis 
(F. Schaudinn), have been advanced to explain the supposed phenomenon, and 
have led to what appears to be much waste of work. We find that the asexual 
forms do not necessarily disappear at all between the relapses, but are generally 
still to be found in small numbers per cubic millimetre on most of the 
days. The apyretic periods preceding the 13 relapses in our cases (7, 17, 18, 
19, 20, 23, 24, 26, 32) lasted 114 days altogether (nearly nine days each on 
the average). On 18 of these days no observations were made ; but on the 
remaining 96 days the parasites were found in small numbers on 59, or 61 per 
cent., and were not found only on 37. Moreover, the general trend of the 
curves suggests that they were not found on these days only because their 
numbers were a little too few for detection. The parasite curve, at its 
height during a pyrexial period, generally falls very rapidly at first and more 
slowly later, and tends to reach its lowest about half-way between two 
apyrexial periods. At this point it may or may not remain above the detect- 
able limit (by thick-film methods). After this it was observed (by D. T.), 
especially in Cases 7, 17, 23, and 24, to begin mounting slowly at first, until, 
when it reached the pyrogenic limit, another pyrexial period commenced. All 
this is scarcely compatible with the speculation that the apyrexial periods 
are due to the abrupt death of most of the asexual Plasmodia, or to their con- 
version into " resting stages," etc. Nor do such speculations appear to be at all 
necessary. It is easy to see that the survival of comparatively small numbers 
of the asexual forms will suffice to keep the infection alive, not only for the 
short periods observed by us, but for " relapses of long interval," and for 
months or years. Obviously, if only a few parasites per cubic milli- 
metre are present, they may easily be overlooked in the small amount 
of blood (say 0*1 cu. mm.) usually examined by thin film, and may 
then be reported as being absent: yet many millions may still exist 



168 Major R. Ross and Dr. D. Thomson. [Oct. 12, 

in a patient who contains, say, 3,000,000 cu. mm. of blood. The 
speculation regarding parthenogenesis in malaria, and the case of 
Schaudinn supposed to support it, have been criticised elsewhere by one of 
us (E. E.).* In two of our relapsing cases (19, 26), no sexual forms at all 
were found during 35 different thick-film examinations. Even if they were 
present in small numbers (crescents do not collect in the spleen), yet the 
numbers of asexual parasites found in or just before the relapses (24 and 400 
per cu. mm.) cannot be explained by parthenogenesis unless we suppose that 
each sexual form produced by " gametoschizogony " 840 or 14,000 spores ! 
Until better evidence for such views is adduced, our results justify the 
doctrine that the malarial infection is kept continuously alive simply by the 
persistence of the asexual forms in varying numbers, and that fever occurs 
only when the forms are numerous enough to produce it. 

9. The Effect of Quinine on the Asexual Forms. — The destructive effect of 
the drug is, of course, everywhere recognised, though very little completely 
satisfactory statistical evidence can be cited in proof. Many laborious 
researches have been made regarding the comparative utility of the various 
salts, but these have been confined almost entirely to estimating the rate and 
percentage of absorption, judged from urinary elimination. The subject is of 
the greatest sanitary and medical importance; but, so far as we can see, it 
can be usefully studied only by the more detailed enumerative analysis 
which we propose soon to undertake. Our daily analysis gives a little 
more light; but the following figures may be mentioned. Cases 15 and 16 
showed no asexual forms; Cases 11 and 13 were so severe that -quinine 
was given at once; Cases 1, 5, 27, 31 and 32 were so mild that quinine 
was withheld during the period of observation ; and in the remaining cases 
the drug was withheld only for some days (except an occasional necessary 
close), and was then given continuously while the parasites were still being 
counted. We have therefore added together all the daily counts during 
the periods of no-quinine and quinine respectively. With eight cases of 
P. vivax there were 45 no-quinine days, showing 104,032 total parasites, or 
an average of 2,312 a clay: and there were 74 quinine days, with 31,769 
total parasites, or an average of only 429 a day (one-fifth). With P. falci- 
parum (19 cases), 147 no- quinine days showed an average of 3,136 asexual 
forms, and 148 quinine days an average of 1,535 (one-half). We should 
have expected that the proportion of quinine-day parasites would have 
been smaller ; but the no-quinine counts are diminished by the inclusion 
of the five mild cases and also by doses of 10 to 20 grains which were 
given on 10 isolated occasions. On quinine days the doses were generally 

* ' Prevention of Malaria ' (Murray, 1910). 



1910.] Some Enumerative Studies on Malarial Fever. 169 

20 to 30 grains a day, and Cases 11, 13, 14, 15 and 16, and parts of other 
cases are omitted because they were unsuitable. Great falls in the number 
of parasites occurred, even without quinine, in Cases 5, 17 and 26 ; while, 
on the other hand, small numbers of asexual forms remained in some cases 
for five or more days in spite of considerable daily dosage. In Case 23 a 
severe relapse began six days after the stoppage of quinine tannate, which had 
been given for nine previous days in 10 to 30 grain doses daily. ISTo other 
relapses occurred after continuous quinine treatment ; but 12 occurred 
without it. 

10. Observations on the Sexual Forms. — It is, of course, generally held — and 
probably quite rightly — that these are developed (by a cy tological process not 
yet clearly seen) from the asexual forms. But, at the same time, no one has 
been able to note any correspondence between their numbers — many of 
the former may be present when the latter are very scarce, and vice versa.. 
This rule, which is fully confirmed by our figures, has always been difficult, 
to reconcile with the accepted theory of origin. On comparing our curves,, 
however, the remarkable fact was observed (by D. T.) that there often seems 
to be indeed a correspondence, but that it is delayed for from 8 to 10 days — 
that is, that the sexual curve tends to rise that number of days after a rise 
in the asexual curve. Out of our 25 infections with P. falciparum 18 
showed crescents, and of these Cases 14, 22, 17, 18, 20, 23, 24 and 30 
suggest this phenomena, while in 17 and 20, the rises are sufficiently 
isolated to show it most distinctly, In Case 17, asexual rises existed on the 
1st to 3rd days, the 11th to 12th and the 21st to 22nd days, separated by 
apyrexial periods during which few asexual and no sexual forms were found ;. 
but on the 30th day (eight days after the last relapse) crescents appeared for 
the first time. In Case 20, an asexual relapse commenced on the 17th day 
and reached its height on the 20th day. A very few crescents had been found 
all the time ; but a marked rise in their numbers commenced on the 26th day 
and reached its height on the 28th day. The most ready explanation is that 
the crescents require 8 to 10 days for development, during which they remain 
undetected, probably in the internal organs of the host. This is confirmed by 
the fact that numerous small ones are seen at the commencement of a rise ;. 
and also by the observation (frequently made) that in fresh infections the. 
crescents seldom appear until a week or more after the first attack of fever. 
Strong confirmation is also given by Case 6, in which Dr. Fantham noted a 
sudden appearance of sexual forms (P. vivax) on the 11th day, 7 to 10 days 
after the original asexual rise, and after the patient had been taking 30 grains 
of quinine daily for a week, suggesting that the same phenomenon holds for 
P. vivax also. 



170 Major R Ross and Dr. D. Thomson. [Oct. 12, 

Other observations are as follows : — (1) By no means every asexual rise is 
followed by a sexual one. In seven cases (13, 19, 25, 26, 28, 31, 33), no 
crescents at all were seen. In Case 13, a negro, 173,400 asexual forms per cubic 
millimetre failed to produce any ; and no crescents were observed in another 
negro (Case 28). (2) The number of crescents never exceeded 5 per cent, of 
asexual forms found in the generation which (hypothetically) produced them, 
and were generally in a much smaller proportion. (3) We could find no 
relation to youth, vigour, amount of haemoglobin, duration of infection (from 
3 to 30 weeks), or season of first infection. (4) The daily counts of crescents 
generally show marked daily variations, but we think that after considerable 
doses of quinine have been given for some days these daily variations tend to 
be smoothed out (Cases 16, 18, 22, 24). (5) In Cases 15, 14 and 16 
particularly, a distinct tertian tendency was observed in the daily variations 
of crescents. This appears not to have been previously noticed, but is just 
what would be expected in the case of a tertian parasite ; though, of 
course, the curve would be confused in a double tertian. (6) Sometimes 
the crescent curve suddenly rises with great rapidity, reaches its maximum, 
and then begins to fall on the next day with equal rapidity ; though, later, 
the fall often tends to be much more slow (Cases 14, 15, 22, 23), 
especially when the numbers are very small. Apparently, the greater the 
maximum the quicker the fall (Cases 14, 15); and the fall is sometimes 
jagged, with a tertian tendency (ibid.). (7) Sometimes the maximum is 
irregularly maintained for a number of days (Cases 16, 18, 24), especially 
when quinine has not been previously given. (8.) The crescents remained 
detectable for as many as 31, 32, 32, 35 and 44 days in Cases 20, 22, 23, 24 
and 18 respectively; and finally disappeared (in 1 cu. mm. of blood) in 
Cases 1, 11, 12, 16, 21 and 23. 

From these data we think (1) that a varying percentage of asexual 
forms are constantly generating crescents, which, after about 8 to 10 days, 
appear in the peripheral blood. Hence, when the asexual forms remain 
sufficiently numerous for a period, as when no quinine is given, the stock 
of crescents is being constantly replenished. Thus the mortality among 
the older crescents is constantly being compensated for by new arrivals, 
and the total numbers appear to remain constant ; an appearance which 
has given rise to the generally accepted hypothesis (perhaps erroneous) that 
the crescents survive for weeks. On the other hand, the sudden rise and fall 
of their numbers seen in Cases 14, 15, 22 and 23 suggest that they really 
survive only for a short time after their appearance in the peripheral blood. 
In Cases 16 and 24, however, a few crescents were still found after quinine 
had been given continuously for 16 to 18 days, suggesting that some of them 



1910.] Some Enumerate Studies on Malarial Fever. 1 7 1 

may be able to survive for longer periods. In Cases 20, 22 and 23, they 
appeared in increasing numbers for 4 to 6 days after continuous 30-grain 
doses of quinine had been commenced, suggesting that the drug has no effect 
upon them when they have once been generated. But quinine appears to 
affect the numbers of crescents indirectly by cutting off the source of supply. 
Though many researches upon the effect of quinine on crescents (a subject of 
the greatest sanitary importance) have been attempted, so far as we know, 
the possibility of a constantly continued supply has not previously been taken 
into consideration. We are continuing researches on the point. 

11. The Leucocytes were counted frequently, but not always daily ; at first 
by the haemocytometer, but in the later 20 cases by the thick-film method. 
The latter method has the advantages of putting the leucocytes closer 
together and of enabling us to estimate . them at the same time with the 
parasites ; but, like the haemocytometer, it is not very appropriate for 
differential counts, because of occasional uncertainty in distinguishing the 
various kinds. Accordingly, these were made (in six cases) by thin film, 
500 cells being examined on each occasion. 

Our results are, as shown in all the cases, that during the pyrexial periods 
the total leucocytes are comparatively few, being frequently as low as 
2000 to 3000 per cubic millimetre (Gases 17, 18, 23, 24, etc.). But as the asexual 
forms and the fever diminish, the total leucocyte curve rises, and about 
seven days later exceeds the normal, and may reach 20,000 and even, rarely, 
30,000 per cubic millimetre (Case 7)— though this height is not maintained. 
The very high counts seem to be associated with much quinine. We observed 
generally that the so-called polymorphonuclear leucocytes, though rather few 
during the pyrexial periods, do not vary much from day to day, but about 
7 days after the fever has abated their numbers are markedly increased 
and vary much. The so-called mononuclear percentage was always in 
excess throughout the cases, even long after the disappearance of all forms 
of the parasites — for six weeks afterwards in Case 1, though quinine had 
been given daily during that period (out of hospital). We think, therefore, 
that a high mononuclear percentage is likely to be always of value in 
diagnosis in the absence of parasites. The normal ratio of total mononuclears 
to polymorphonuclears is about 35 per cent., but in our cases it was often 
60 per cent, and in Case 30 reached 80 per cent. It tends to fall slightly 
with great improvement in health. During the individual daily paroxysms, 
as shown by Stephens and Christophers, the total mononuclears are rather 
low during the height of the fever, but show a marked increase during the 
following remission. This increase is chiefly due to the large mononuclears. 
This process is repeated with each paroxysm, and if a paroxysm fails the 



172 Major R. Ross and Dr. D. Thomson. [Oct. 12, 

mononuclears remain (Case 9 especially). But we could not find these 
variations in the absence of fever and many parasites. This mononuclear 
reaction, which we have also seen in our case of trypanosomiasis, appears 
to he exactly comparable to the polymorphonuclear reaction described by 
F. W. Andrewes* as generally occurring in bacterial diseases. 

12. Various Therapeutic Agents. — Methylene blue was tried in three cases 
in 12 -grain daily doses ; the parasites diminished, but we cannot draw any 
conclusions from the figures. In Case 24, altogether 36 grains of soamine 
were injected intramuscularly in 5 to 10 grain doses on five occasions during 
12 days, and yet a smart relapse occurred during and after the last dose. In 
the same case, just before the same relapse, X-rays had been applied over 
the spleen and abdomen for 20 minutes. The asexual parasite rise had 
commenced before the application. In Case 24, faradic and galvanic currents 
were applied over the spleen, each for 10 minutes, but crescent counts made 
immediately before and two hours after the application remained unaltered. 

13. Hcemoglobin. — This was estimated in most of the later cases. The 
percentage always fell during fever, and began to rise rapidly shortly after- 
wards, proving the destructive effect of the parasites and the rapid recupe- 
ration of the patients. The greatest fall was 25 per cent, after four days' 
fever (Case 20) ; but more detailed work is required to trace correspondence 
with the number of parasites. The lowest percentage found was 53 per cent. 
All the patients were anaemic at first, but the haemoglobin rose rapidly with 
improvement of health, and very rapidly in the more vigorous subjects. We 
decided not to attempt counts of red corpuscles, as these have been made so 
frequently already. The elimination of the haemoglobin and the findings in 
a case of blackwater fever will be dealt with in the following paper by 
Dr. Simpson and in one by ourselves.*!* 

14. Summary. — (1) There is a very decided correlation between the number 
of asexual Plasmodia found in the peripheral blood and the fever. 

(2) As a rule, no fever exists unless the asexual forms exceed some 
hundreds per cubic millimetre. 

(3) The asexual forms do not always disappear between relapses (as often 
thought) but tend to persist in small numbers per cubic millimetre, and often 
increase again for some days before the actual febrile relapse occurs. 

(4) These observations give a coherent theory of the malarial invasion, 
according to which the infection is kept alive indefinitely by the ordinary 
sporulation of the asexual forms, and not by parthenogenesis or by resistant 

* ' Lancet,' June, July, 1910. 

t ' Annals of Tropical Medicine,' Liverpool, vol. 4, No. 3. 



1910.] Some Enumerative Studies on Malarial Fever. 173 

forms : and fever recurs only when the parasites are numerous enough to 
produce it. 

(5) We estimate from our cases that considerable continued doses of 
quinine reduced the asexual forms by 50 to 80 per cent. 

(6) There are strong reasons for supposing that the sexual forms require 
eight to ten days for development ; that the often noticed long persistence of 
crescents is not due to their long life (as generally thought) but to constant 
replenishment of the stock by fresh broods ; that they sometimes show a 
distinct tertian periodicity ; and that quinine does not affect them when once 
generated, but ultimately reduces their numbers by destroying the generating 
cells. The sexual forms were never seen to produce fever. 

(7) The leucocytes are below normal during febrile periods and above 
normal afterwards. The percentage of mononuclears rises greatly after 
paroxysms, and is always in excess of the normal. 

(8) Methylene blue, soamine, X-rays, and faradic and galvanic currents had 
no results in a few experiments. 

(9) The haemoglobin falls markedly with fever, but rises rapidly w r ith 
convalescence. 

(10) The fa3cal urobilin shows marked correlation with the occurrence of 
fever, and is specially studied in the accompanying paper by Dr. Gr. C. E. 
Simpson.* 

Many other deductions may be based on our observations, but will be 
better considered after more detailed counts have been made. 

* ' Koy. Soc. Proc.,' B, vol. S3, p. 174. 



VOL. LXXXIII. — B.